Target details

Target details

ShowHide

Background

Factor V (FV) is an essential cofactor of the blood coagulation cascade and circulates in plasma as a large single-chain glycoprotein (330 kDa). The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. During coagulation, it is converted to the active cofactor FVa via limited proteolysis by thrombin, and spliced into a heavy chain (110 KDa) and a light chain (73 KDa) held together non-covalently by calcium. In the presence of a calcium ion and the phospholipid on cell surfaces, FVa and Fxa form the prothrombinase complex which catalyzes the hydrolysis of prothrombin to thrombin. Thrombin in turn cleaves fibrinogen to fibrin which polymerizes to form a clot. FVa is readily inactivated by anticoagulant activated protein C. FV Leiden, a single amino acid mutation, renders FVa resistant to cleavage by activated protein C. It therefore over-produces thrombin, and leads to excess clotting and hereditary thrombophilia disorder. Severe FV deficiency is associated with mild to severe bleeding diathesis.

Research Area

Coagulation

Application Details

Application Details

ShowHide

Sample Volume

50 μL

Assay Time

< 4 h

Plate

Pre-coated,Strips (12 x 8)

Protocol

This assay employs a quantitative sandwich enzyme immunoassay technique that measures Factor V in less than 4 hours. A polyclonal antibody specific for Factor V has been pre-coated onto a microplate. Factor V in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for Factor V, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated Factor V Antibody to each well and incubate for one hour. Wash the microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 20 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calculation of Results

Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot 4-parameter graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. T The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision

Intra-assay and inter-assay coefficients of variation were 4.8 % and 7.4% respectively.

Restrictions

For Research Use only

Handling

Handling

ShowHide

Handling Advice

Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated-antibody vial before opening and using contents. The kit should not be used beyond the expiration date.

Storage

4 °C/-20 °C

Storage Comment

Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent